Cerebrovascular disorders (the term "cerebrovascular disorders" as used herein means disorders in various brain cells and intracerebral blood vessels induced by, for example, ischemia) break out due to so-called cerebral ischemia, wherein the blood stream in the brain is lowered to the threshold or below, which is induced by occlusion of blood vessel caused by a constriction of blood vessel, cerebral thrombosis or cerebral embolus. Kirino et al. [Brain Res., 377:344-347 (1982)]found out that when transient cerebral ischemia was loaded to a gerbil, so-called delayed neuronal cell death, i.e., slow and delayed hippocampal pyramidal neuronal cell death was observed 2 or 3 days thereafter. Although attempts have been made to clarify the degenerative process of neuron after central damage (for example, ischemia), this mechanism has not been clarified so far.
According to Siesjoe and Bengtsson [J. Cereb. Blood Flow Metab., 9:127 (1989)], at ischemia, glutamate is liberated from the presynaptic side to synaptic clefts and then binds to a glutamate receptor in the postsynaptic side. Thus the calcium ion influx into the cells and the liberation of calcium ion from intracellular storage sites are promoted. At the same time, the extrusion of intracellular calcium ion is suppressed due to decreased calcium-ATPase activity. As a result, the intracellular calcium ion concentration is elevated, which results in the neuronal cell death. Compared with the extracellular calcium ion concentration, the intracellular calcium ion concentration is extremely low. It is known that cells cannot survive when the intercellular calcium ion concentration is elevated to a certain level. However the process from an increase in the intracellular calcium ion concentration to neuronal cell death has not been clarified yet.
Also, there have been reported that at cerebral ischemia, calmodulin, which is a calcium-binding protein, was activated [Picone et al., J. Cereb. Blood Flow Metab., 9:805-811 (1989)], the activity of calmodulin-dependent protein kinase was changed [Churn et al., Stroke, 21:1715-1721 (1990)]and breakdown of fodrin, which is a calmodulin-binding cytoskeltal protein, was accelerated by cerebral ischemia disorders [Seubert et al., Brain Res., 492:366-370 (1989)].
On the other hand, there have been also reported that calmodulin accelerated the breakdown of fodrin by calpain [Harris et al., J. Biol. Chem., 264:17401-17408 (1989)]and trifluoropelazine, which is a compound having a calmodulin-inhibition effect, suppressed the breakdown of fodrin [Seubert et al., Synapse, 1:20-24 (1987)]. However it has never been reported that these phenomena participate in neuronal cell death.
Although it is considered that a drug having a calmodulin-inhibition effect might be applicable to antihypertensive drug, antianginal drug, antiarrhythmic drug, drug for treating schizophrenia or drug for improving cerebral circulation on the basis of vasodilator effect, no effect of suppressing neuronal cell damage has been proved so far. Kogure et al. reported that as a result of their examination, W-7, which is a substance having a calmodulin- inhibition effect, exhibited no effect of suppressing delayed neuronal cell death and thus denied the applicability of a calmodulin inhibitor as a drug for treating cerebrovascular disorders (Kogure et al., Tanpakushitsu, Kakusan, Koso, 35:1254 (1990)].
It has been reported that a phenothiazine compound, which also has an effect of inhibiting calmodulin, relieved cerebral ischemic disorders by its antioxidant effect [Ye et al., Stroke, 23:1287-1291 (1992)].
With the tendency toward an aging society in recent years, increases in brain disorders including cerebrovascular disorders and Alzheimer's disease have become a serious social problem. It is known that the root of these diseases lies in brain neuronal cell death caused by various factors. For example, cerebrovascular disorders are induced by cerebral ischemia and the severity of these diseases relates to the ischemic period. Slight ischemia brings about little problem, while prolonged ischemia results in irreversible damages in the brain. Since matured neurons are not regenerated through cell division any more, these disorders remain as permanent organic changes and strongly affect the prognosis.
Therefore, it is highly useful in treating cerebrovascular disorders and relieving the sequelae thereof to suppress neuronal cell death. Further, it has been proposed that neuronal cell death based on the accelerated degradation of cytoskeltal protein causes Alzheimer's disease. Accordingly, suppression of neuronal cell death is useful in the treatment and prevention of Alzheimer's disease and relief of the sequelae thereof too.
If the excessive activation of calmodulin induced by an increase in the intracellular calcium ion concentration at neuronal cell disorders can be suppressed by using a calmodulin inhibitor, therefore, neuronal cell death can be suppressed.
Delayed neuronal cell death occurs several days after the break out of cerebrovascular disorders. The present inventors have found out that the content of calmodulin in cytoplasm begins to decrease from the early stage (more concretely, even 1 hour after ischemia) while the calmodulin content in membrane fraction increases. This is the same phenomenon as the one observed when an excessive amount of calcium ion is added to a hippocampal homogenate. It indicates that calcium ion binds to calmodulin at cerebral ischemia and some part thereof translocates into the membrane side. The present inventors have further clarified that the degradation of fodrin, which is a cytoskeltal protein contained in the membrane, is accelerated by the addition of calcium ion and that calmodulin binds to the breakdown products.
The present inventors have further found out that in a cerebral ischemic model of gerbil, the breakdown of fodrin is accelerated prior to neuronal cell death. Thus they have examined compounds A and B, each having a high selectivity for calmodulin and an intense calmodulin inhibition effect, and consequently found out that these compounds suppress the translocation of calmodulin into the membrane in the early stage of cerebral ischemia, suppress the breakdown of fodrin and, in its turn, suppress neuronal cell death. The present invention has been thus completed.